Methods for preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample and compositions for practicing the same

Abstract

Methods of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample are provided. Aspects of the methods include combining the RNA sample with a first strand cDNA primer and a template switch oligo-nucleotide under first strand cDNA synthesis conditions, where one of the first strand cDNA primer and the template switch oligo-nucleotide includes a first post-tagmentation amplification primer binding domain. The resultant product is subjected to amplification conditions sufficient to produce a double stranded cDNA, which is then tagmented with a transposome that includes a second post-tagmentation amplification primer binding domain. The tagmented sample is then subjected to amplification conditions using first and second post-tagmentation amplification primers that include sequencing platform adapter constructs to produce a NGS library. Aspects of the invention further include compositions produced by the methods and kits that find use in practicing the methods.

What is claimed is: 
     
       1. A method of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample, the method comprising:
 (a) combining:
 a RNA sample; 
 a first strand complementary deoxyribonucleic acid (cDNA) primer comprising a first pre-tagmentation amplification primer binding domain; 
 a template switch oligonucleotide comprising a 3′ hybridization domain and a 5′ second pre-tagmentation amplification primer binding domain; 
 a reverse transcriptase; and 
 dNTPs; 
 
 in a reaction mixture under conditions sufficient to produce a double stranded product nucleic acid comprising a template mRNA and the template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein only one of the first strand cDNA primer and the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain; and 
 (b) contacting the product nucleic acid with first and second pre-tagmentation amplification primers complementary to the first and second pre-tagmentation amplification primer binding domains under amplification conditions sufficient to produce a product double stranded cDNA; 
 (c) tagmenting the product double stranded cDNA with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon end domain and a second post-tagmentation amplification primer binding domain to produce a tagmented sample; and 
 (d) amplifying the tagmented sample by contacting the tagmented sample with:
 a first post-tagmentation primer comprising a first post-tagmentation amplification primer domain complementary to the first post-tagmentation amplification primer binding domain; and 
 a second primer comprising a second post-tagmentation amplification primer domain complementary to the second post-tagmentation amplification primer binding domain. 
 
 
     
     
       2. The method according to  claim 1 , wherein
 the first post-tagmentation primer further comprises a first NGS indexing domain and a first NGS adapter domain; and 
 the second post-tagmentation primer further comprises a second NGS indexing domain and a second NGS adapter domain. 
 
     
     
       3. The method according to  claim 1 , wherein the RNA sample comprises messenger RNA and the method comprises producing the NGS library from mRNA. 
     
     
       4. The method according to  claim 1 , wherein the first strand cDNA primer comprises the first post-tagmentation amplification primer binding domain. 
     
     
       5. The method according to  claim 1 , wherein the template switch oligonucleotide comprises the first post-tagmentation amplification primer binding domain. 
     
     
       6. The method according to  claim 1 , wherein the first and second pre-tagmentation amplification primer binding domains are identical and the first and second pre-tagmentation amplification primers are identical. 
     
     
       7. The method according to  claim 1 , wherein the transposase comprises a Tn5 transposase. 
     
     
       8. The method according to  claim 7 , wherein the transposon end domain comprises a Tn5 transposon end domain. 
     
     
       9. The method according to  claim 1 , wherein the method further comprises pooling the double stranded product cDNA with a second double stranded product cDNA to produce a pooled cDNA sample, and then tagmenting the pooled cDNA sample. 
     
     
       10. The method according to  claim 1 , wherein the RNA sample is one that is produced from a single cell. 
     
     
       11. The method according to  claim 1 , wherein the method further comprises subjecting the NGS library to an NGS protocol. 
     
     
       12. The method according to  claim 1 , wherein the method further comprises quantitating one or more RNA species of the RNA sample. 
     
     
       13. The method according to  claim 4 , wherein the first strand cDNA primer comprises an oligodT domain 3′ of the first pre-tagmentation amplification primer binding domain. 
     
     
       14. The method according to  claim 1 , wherein the first strand cDNA primer further comprises a source barcode domain. 
     
     
       15. The method according to  claim 1 , wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs. 
     
     
       16. The method according to  claim 15 , wherein each of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs. 
     
     
       17. The method according to  claim 15 , wherein the one or more nucleotide analogs is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, linkage modifications, 5′ and/or 3′ end modifications, one or more fluorescently labeled nucleotides, and combinations thereof. 
     
     
       18. The method according to  claim 17 , wherein the one or more nucleotide analogs comprises a 5′ end modification. 
     
     
       19. The method according to  claim 1 , wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation primers comprises a 5′ polymerase blocking modification. 
     
     
       20. The method according to  claim 19 , wherein each of the first strand cDNA primer and pre-tagmentation primers comprises a 5′ polymerase blocking modification. 
     
     
       21. The method according to  claim 19 , wherein the 5′ polymerase blocking modification is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, and combinations thereof. 
     
     
       22. The method according to  claim 1 , wherein the 3′ hybridization domain comprises a homonucleotide stretch. 
     
     
       23. The method according to  claim 1 , wherein the 3′ hybridization domain comprises a heteronucleotide stretch.